The enzymatic reduction of atmospheric dioxygen, O2, is an event of prime biological importance. The complete reduction of O2 to two molecules of water requires, in sequence, four electrons in an overall process responsible for virtually all energy production from the oxidation of common food stuffs. An equally important reaction is the two electron reduction of O2 to the redox level of peroxy anion, O2, exemplified by mixed function oxidases, such as the P450 hydroxylase systems which cleave the oxygen-oxygen bond reducing via two electrons, one atom to water while the second formally oxidizes a substrate via oxygenation. These reactions play a crucial role in steroid metabolism in the adrenal cortex, and detoxification and solubilization of environmental toxins and carcinogens in the liver. The P450 hydroxylase system under study in our laboratory is isolated in pure and homogeneous form from the soil bacterium Pseudomonas putida, and serves as an excellent model for studying the electron transport and catalytic events of these important enzymes. We have defined the physical and chemical properties of the isolated P450 cytochrome, and the flavoprotein reductase and iron-sulfur redoxin that shuttle electrons from the biochemical reducing agent NADH to the cytochrome. Current investigation centers on the interaction of protein components and the selective and specific modification of these species in order to elucidate the molecular mechanisms of the transfer of redox equivalents and subsequent hydroxylation of substrate. BIBLIOGRAPHIC REFERENCES: Gunsalus, I.C., Pederson, T.C. and Sligar, S.G. Oxygenase-Catalyzed Biological Hydroxylations. In Annual Review of Biochemistry 45, 377-407. 1975. Sligar, S.G., Debrunner, P.G., Namtvedt, M.J. and Gunsalus, I.C. Multienzyme Associations During Mixed Function Oxidation by Cytochrome P450 cam and Putidaredoxin. Fed. Proc. 34,622. 1975.